This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The dynamic modification of intracellular proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) is a critical and ubiquitous cell signaling paradigm, regulating substrates'function, localization and stability. Protein OGlcNAcylation controls numerous processes in a wide range of mammalian tissues, and its dysregulation is implicated in human diseases, such as type II diabetes and neurodegeneration. Despite its broad functional significance, major aspects of O-GlcNAc signaling are poorly understood. Specifically, three key questionsremain unanswered: 1. What are the functionally important substrates in pathways that O-GlcNAc regulates? 2.What biochemical effect does O-GlcNAc have on these substrates? 3. How are proteins targeted for signal dependent O-GlcNAcylation? Because O-GlcNAc is a transient post-translational modification not under direct genetic control, these questions are challenging to answer using traditional molecular biology techniques alone. Therefore, we have pioneered chemical biology approaches to studying O-GlcNAc that make use of unnatural sugar reagents and cognate detection probes. We will apply these tools to study the role of O-GlcNAc in regulating two model cell biological processes: the DNA damage response and mitochondrial metabolism. Importantly, the identification of glycosylation sites on O-GlcNAc targets is critical for understanding the role of the modification in both processes. The UCSF Mass Spectrometry Facilty will provide vital expertise and collaborative resources for identifying O-GlcNAcylation sites on proteins of interest. Together, the project will exploit both novel chemical methods and mass spectrometry to dissect the functional role of O-GlcNAc in mammalian cell physiology.